PROJECT SUMMARY ? CORE C The ACCESS-GI (Advanced Co-culture Engineering and Single cell Statistics of Gut Immunology) Core C will provide a range of cutting edge approaches to Stanford NAMSED investigators (Projects 1, 2 and pilots) to analyze intestinal/gastric epithelial cells and their interactions with immune cells both with and without infection. In association with the Organoid Core B, we will carry out development work towards the generation of organoids that integrate mucosal immune cells. Specifically, in Aim 1, we will leverage highly multiplexed, single cells analysis (CyTOF mass cytometry) and imaging (multiplexed ion beam imaging ? MIBI) modalities collaboratively developed at Stanford by Sean Bendall to provide cell analysis assays for projects across the NAMSED center (Projects 1, 2). These assays will be uniquely beneficial, allowing the simultaneous characterization of human gut organoid structure, the integration of immune cells, and the effects of pathogens on cellular behavior. Further to this, Core C will facilitate access to other analysis modalities available through Stanford's human immune monitoring core (HIMC), including 63-plex Luminex for quantitative measurement of human cytokines and chemokines, standard ELISA for non-Luminex targets, and Fluidigm Biomark for single cell qPCR analysis. The core will leverage the expertise of Sean Bendall, his team, and the HIMC for analysis of data generated by these platforms and, where appropriate, for multi-platform data integration. In Aim 2, Sarah Heilshorn and her team will provide novel assays to NAMSED investigators (Projects 1, 2), using microfluidic devices that allow investigation of cell/cell and cell/pathogen interactions in highly controlled micro-environments. In Aim 3, Elizabeth Mellins and her team, in collaboration with the Organoid Core, will provide M-cell-like organoids to Project 1, and class II expressing organoids and develop production of organoids with physiologic levels of M cells. Her team will also provide homogenous immune cell populations to NAMSED investigators for experiments addressing immune reactivity in the context of infected intestinal organoids (Project 1) as well as study interactions of selected antigen presenting cells with uninfected and infected organoids. The latter will form the foundation of a key effort, in collaboration with the Organoid Core, to develop human intestinal organoids that more closely replicate the physiologic microenvironment encountered by pathogens during enteric infection.